Extraction of vitamin b12-active compounds from aqueous solutions using solid trichlorophenol



Patented Apr. 21,, 1953 EXTRACTION OF- VITAMIN BIQrACTIVE M,-

POUNDS FROM AQUEOUS SOLUTIONS IN SOLID RICHL OB E QL Robert G.Dcnkewalter, Westfield, George B, and; Stephen Kutosh, Kenilworth, N; .1assignors to Merck & 00., Inc., Rahway, J., a corporation of New:

Hughey, Metuchen,

Jersey NoDr-aw ing, ApplicationiAugnst 23, 1950,

Serial No. 181,106

4 Claims. 1.

This invention, is concerned generally withthe recoveryofanti-pernicious anemia, factors from aqueous solutions, Moreparticularly, it relates tofa novel process for separating vitamin B12and vitamin B1 -like compounds from aqueous solutions, inc1udingfermentation broths, containing the same, by extracting the vitamin B12and/or vitamin B n-lik compounds from said aqueous solutions utilizingsolid 2,4,5-trich1oropheno1 as the extracting agent.

Vitamin B is a new chemical compound fully characterized in a co-pendingapplication of applicants assignee, SerialNo. 146,4091, filed February25,1950, and" in U. S. Patent No. 2,563,794, Whivhis capa e o n motina eg w f h microorganism Lactobacz'llus lactic, Burner, and which possessesmarked and effective action in the therapeutic treatment of Addisonianpernicious. anemia and. other macrocyticanemias. VitaminABm isordinarily prepared byfermenting anaqueousnutrient medium by means of avitamin. Biz-producing strain of: Streptomyces and subjecting thefermentation broth thus obtained to a purification operation.

By, vitamin Biz-like i compounds aremeant the red. crystallineocompounds (not vitamin B12) which are obtained when a fermentation brothcontaining them 1 is subjected to 1 the 1 purificationtreatmentiutilizediin the purification of 1 vitamin B12, (as describedon pages 13 to 1 6 ofsaid application Serial No. 1 16;404) but omittingthe counter-current distribution procedure employed as the final step 1in; this purification operation. Theseevitamin B12-1ike compounds can becharacterized byv the fact that they are readily convertible to purevitamin BIZ'DEI seiby-treatmentwith cyanide ion, as described inaco-pending-application of applicants assignee, SerialNm 120,009,filedfictober 6, 1949; now Patent No. 2,530,416 issued November 21,1950. Vitamin Biz-and vitamin.,B12- -1ike..compounds can be referred=toby the/expression, vitamin B12active-compounds= The, recoverypf avitamin Biz-active compound, that: is vitamin B12 and/or a vitaminB12-1ike compound, from, Streptomyces: fermentation brothshasheretofore-been accomplished by treating said ibrothsiwith an=adsorbentmaterial such a e-activated ,charcoal sorefullerzs earth which 'producesanadsorbate containing vitamin B12 and/0r vitamin Biz-like compoundspresent inthe-broth togetheriwith various-contaminants. Antibiotics,suchv as streptomycintor grisein, which may be present, in said brothareadsorbed along with the,vit .i .1.,-- 3 2, and/or. .1 vitamin Biz-miccompounds. The adsorbate is then eluted with a suitable solvent, such;as an aqueous solution of pyridine or an alkyl substituted pyridine, andthe eluate evaporated thereby producing an elaboration product whichcontains the vitamins B12 and/or vitamin B -Iike compounds, togetherwith some of the contaminants, such as grisein or streptomycin, whichmay have been present in the original broth. The elaboration productthus obtained is extracted with an elaboration solvent, such as wateroralower-aliphatic watermiscible alcohol, and the resulting extract issubjected to chromatographic fractionation by adding said extract to acolumn of adsorbentmaterial and washing thecolumn; with;solvent,fractionally to elute the vitamin B12 andyor vitamin B12 compounds fromthe adsorbent material.

The resulting eluate can then be further processed asset forth in theaforementionedapplication Serial No. 146,404 and in U. S. Patent No.2,563,794, to produce pure vitamin B12, or to produce the vitaminB12-like substances substantially free of contaminating materials,

Thus, the preparation of a vitamin enriched extract suitable forchromatography, starting with a fermentation broth containing vitaminB12 and/or vitamin B12-like compounds, previously involved the followingpurification operations: (1) adsorption of the vitamin B12 and/orvitamin B12-1ike substances from fermentation broths utilizing activatedcarbon or fullers earth as the adsorbing agent; (2) elution of'theadsorbate with pyridine; (3) evaporation of the pyridine eluate; and (4)extraction oi the elaboration product With another solvent.

It is now discovered that the preparationof a vitamin enriched extractsuitable for chromatography can be, accomplished in a single adsorptionand elution operation starting with a fermentation broth containingvitamin 1312 and/or vitamin BIZ-like compounds, This is accomplished byutilizing, instead 'of the adsorbents heretofore employed for, thispurpose, a solid extracting agent possessingsuperiorselectiveproperties. This superior extractant is solid 2;4, 5trichlorophenol. When anaqueoussolution containing vitamin B 2 orvitamin Biz-like compounds is mixed with' or passedthrough a columnofsolid2;l,5=trichlorophenol; the vitamin B12 and/or -v-itam-in B12-1ikesubstances are substantially quantitatively extracted from the aqueoussolution; Moreover, whenafermentation broth containing vitamin B12and/or vitamin B12-1ike compounds is-contactedwith solid2,4,5-trichloropheno1; it has been-found that the trichlorophenolselectively extracts vitamin B12 wereas the and/or vitamin Bm-likecompounds, while, at the same time, leaving behind in the residual brothcontaminants which are ordinarily adsorbed utilizing conventionaladsorbing agents such as activated carbon or fullers earth.

It is a further feature of this invention that the solid2,4,5-trichlorophenol extract (1. e. containing extracted vitamin B12and/or vitamin B12-like compounds) can be dissolved in a suitablesolvent to form a solution of the vitamin materials. The vitamin B12 andvitamin Biz-like substances are readily extracted from this solution bymeans of water, using either a batch extractor or an extraction column,whereby substantially quantitative transfer of vitamin B12 and vitaminB12- like substances to the water phase is secured. This aqueousextraction step is accompanied by a further separation and purificationof vitamins since some of the contaminating materials are retained inthe non-aqueous layer. This procedure thus combines the advantages ofutilizing a solid extracting material together with the advantages of aliquid-liquid extraction operation for the recovery of the extractedcomponents.

The fact that 2,4,5-trichlorophenol can be employed in solid form isvery unusual since other phenolic compounds cannot be used for thispurpose. For example, various phenolic compounds such as p-octylphenol,pentachlorophenol, ophenyl-phenol, and the like, as well as2,4,5-trichlorophenol itself, when employed in liquid form (1. e. at atemperature above the melting point where the phenolic compound is asolid at room temperature), will extract vitamin B12 and/or vitaminB12-like compounds from aqueous solu tions. No other phenolic compound(such as, for example the p-octylphenol, the pentachlorophenol, or thep-phenyl phenol enumerated above) has been found to effect measurableextraction of vitamin B12 and/or vitamin B12- like substances fromaqueous solutions when the phenolic compounds are utilized in the solidform. It is indeed surprising, therefore, that, when2,4,5-trichlorophenol is employed as a solid, it accomplishes thisextraction in a rapid and quantitative manner to form a solid solutionof vitamin B12 and/or vitamin B12- lilre compounds in the phenolicextractant.

It is a special feature of this invention that 2,4,5-trichlorophenol canbe readily' prepared in the form of small hard pellets, particularlyadvantageous for column operation. Other related phenols are frequentlysoft and oily and obtainable as a solid only in larger and irregularshapes.

The advantages of utilizing solid 2,4,5-trichlorophenol as an extractionagent, of course, obviates the emulsion difiiculties frequentlyencountered in liquid-liquid extraction procedures and there is noseparation problem. A further feature of this extraction utilizing theextracting agent in solid form is that the quantity of solid phenolicextractant is very much less than that required in a liquid-liquidextraction operation, and that the loss of extractant is very muchreduced as contrasted with the losses involved when a liquid extractantis utilized. This is particularly true in column operation, where, forexample, it is possible, utilizing solid 2,4,5-trichlorophenol, toeffect a complete extraction of vitamin B12 and/or vitamin B12-likesubstances from an aqueous solution utilizing one part of phenoliccompound to 500 parts of solution,

solvent requirement for a liquidtion is approximately 1 part of ex-.parts of the solution.

In carrying out the present invention, an aqueous solution containingvitamin B12 and/or vitamin B12-like substances is mixed with or passedthrough a column of solid 2,4,5-trichlorophenol, whereby the vitamin B12and/or vitamin Bm-like substances are extracted by the phenoliccompound.

The 2,4,5-trichlorophenol extract is then contacted with water and anorganic solvent immiscible with water, (in which 2,4,5-trichlorophenolis soluble and in which vitamin B12 and/or vitamin B12-like compoundsare substantially insoluble), such as a chlorinated hydrocarbon, forexample carbon tetrachloride, chloroform, ethylene dichloride and thelike. Alternatively, the solid 2,4,5-trichlorophenol can first bedissolved in an organic solvent miscible with water (forming a solutionin which both 2,4,5-trichlorophenol and vitamin B12 and/or vitaminB12-like compounds are soluble) such as acetone, methanol and otherlower aliphatic alcohols. Water, and an organic solvent of the typefirst mentioned, can then be added to the organic solvent solutioncontaining the 2,4,5-trichlorophenol and the vitamin B12 and/or vitaminB121ike compounds. The water layer which results from either of theforegoing procedures contains substantially all of the vitamin B12and/or vitamin B12-like compounds; this water layer is separated and canbe further processed as described in the co-pending application ofapplicants assignee mentioned hereinabove, namely Serial No. 146,404,and in U. S. Patent No. 2,563,794, to produce pure vitamin B12.

The following example illustrates a method of carrying out the presentinvention, but it is to be understood that this example is given forpurposes of illustration and not of limitation.

Example A fermentation broth, obtained by fermenting an aqueous nutrientmedium with a strain of Streptomyces grz'seus which produces bothgrisein and vitamin B12 substantially as described in the co-pendingapplication of applicants assignee, Serial No. 146,404, was clarified byfiltration and adjusted to a pH of 4.0 by the addition of hydrochloricacid. This broth was passed through a column packed with solid spheresof 2,4,5-trichlorophenol, which column was prepared as follows: 6000gms. of 2,4,5-trichlorophenol was melted and formed into small solidspheres by pouring into agitated cold water. These spheres of solidtrichlorophenol were placed in a glass column 3 inches in diameter and15 feet in height, equipped with feed and drain connections. The brothwas passed through the column in upward flow at a rate of 700-800 mL'perminute until the entire 225 gallons had passed through. The effluent wasdiscarded. The column was then washed with water to remove residualbroth. i

The trichlorophenol adsorbate was next dissolved by adding 3 liters ofacetone. To the resulting solution was added 3 liters of water and 36liters of carbon tetrachloride. The aqueous layer was separated, theorganic layer was extracted with 3 liters of water, and the water layerswere combined. Carbon tetrachloride was removed from the organic layerby distillation, and the residual 2,4,5-trichlorophenol was saved I01re-use.

The combined water layers were saturated with ammonium sulfate and thenextracted twice with 300 ml. of benzyl alcohol. The combined benzylalcohol extracts were chromatographed, precipitated, and further treatedsubstantially as described in the co-pending application of applicantsassignee Serial No. 146,404, filed February 25, 1950, and in U. S.Patent No. 2,563,794, thereby producing pure vitamin B12.

Various changes and modifications may be made in carrying out thepresent invention without departing from the spirit and scope thereof.Insofar as these changes and modifications are Within the purview of theannexed claims, they are to be considered as part of our invention.

We claim:

1. In a process for recovering an anti-pernicious anemia factor,consisting of a vitamin B12- active compound, from an aqueous solutioncontaining said factor, the step which comprises contacting saidsolution with solid 2,4,5-triohlorophenol thereby extracting said factorfrom said aqueous solution.

2. In a process for recovering an antipernicious anemia factor,consisting f a vitamin Bm-active compound, from a fermentation brothcontaining said factor, the steps which comprise contacting said brothwith solid 2,4,5-trichlorophenol thereby extracting said factor fromsaid broth, and bringing the trichlorophenol extract thus obtained intointimate contact with water and a water-immiscible organic liquidcomprising a chlorinated hydrocarbon solvent, thereby forming atwo-phase mixture consisting of a nonaqueous layer containing thetrichlorophenol and a water layer containing the anti-pernicious anemiafactor.

3. In a process for recovering vitamin B12 from a fermentation brothcontaining that vitamin, the steps which comprise contacting said brothwith solid 2,4,5-trichlorophenol thereby extracting said vitamin B12from said broth, and bringing the trichlorophenol extract into intimatecontact with water and a water-immiscible organic liquid comprisingcarbon tetrachloride, thereby forming a two-phase mixture consisting ofa non-aqueous layer containing the trichlorophenol and a water layercontaining the vitamin B12.

4. In a process for recovering vitamin B12 from a fermentation brothcontaining that vitamin, the steps which comprise contacting said brothwith solid 2,4,5-trichlorophenol thereby extracting the vitamin B12 fromsaid fermentation broth, dissolving the resultingtrichlorophenol-vitamin B12 extract in acetone, and contacting theacetone solution thus obtained with water and carbon tetrachloride,thereby forming a two-phase mixture consisting of a carbon tetrachloridelayer containing the 2,4,5-trichlorophenol and an aqueous layercontaining the vitamin B12.

ROBERT G. DENKEWALTER. GEORGE B. HUGHEY. STEPHEN KUTOSH.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,134,256 Laland Oct. 25, 1938 2,530,416 Wolf Nov. 21., 1950FOREIGN PATENTS Number Country Date 349,794 Germany Mar. 9, 1922 OTHERREFERENCES Smith: Proceedings of the Biochemical Society in BiochemicalJournal, volume 43 (Number 1), October 1948, pages VIII, IX.

Fantes: Proceedings of the Royal Society (1950) pages 592 to 596.

1. IN A PROCESS FOR RECOVERING AN ANTI-PERNICIOUS ANEMIA FACTOR,CONSISTING OF A VITAMIN B12ACTIVE COMPOUND, FROM AN AQUEOUS SOLUTIONCONTAINING SAID FACTOR, THE STEP WHICH COMPRISES CONTACTING SAIDSOLUTION WITH SOLID 2,4,5-TRICHLOROPHENOL THEREBY EXTRACTING SAID FACTORFROM SAID AQUEOUS SOLUTION.